Pneumococcal and meningococcal vaccines formulated with interleukin-12

ABSTRACT

This invention pertains to vaccine compositions comprising a mixture of antigen, such as pneumococcal or meningococcal antigen, and interleukin IL-12, which may be adsorbed onto a mineral in suspension. The pneumococcal or meningococcal antigen may be conjugated to a carrier molecule. These vaccine compositions modulate the protective immune response to the antigen.

[0001] This application is a divisional of U.S. application Ser. No.09/248,195, filed Feb. 10, 1999, the entire disclosure of which ishereby incorporated by reference, which claims the benefit of U.S.Provisional Application No. 60/074,528, filed Feb. 12, 1998, the entiredisclosure of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] The immune system uses many mechanisms for attacking pathogens;however, not all of these mechanisms are necessarily activated afterimmunization. Protective immunity induced by vaccination is dependent onthe capacity of the vaccine to elicit the appropriate immune response toresist or eliminate the pathogen. Depending on the pathogen, this mayrequire a cell-mediated and/or humoral immune response.

[0003] The current paradigm for the role of helper T cells in the immuneresponse is that they can be separated into subsets on the basis of thecytokines they produce, and that the distinct cytokine profile observedin these cells determines their function. This T cell model includes twomajor subsets: TH-1 cells that produce IL-2 and interferon γ (IFN-γ)which augment both cellular and humoral immune responses, and TH-2 cellsthat produce IL-4, IL-5 and IL-10 which augment humoral immune responses(Mosmann et al., J. Immunol. 126:2348 (1986)). It is often desirable toenhance the immunogenic potency of an antigen in order to obtain astronger immune response in the organism being immunized and tostrengthen host resistance to the antigen-bearing agent. A substancethat enhances the immunogenicity of an antigen with which it isadministered is known as an adjuvant. For example, certain lymphokineshave been shown to have adjuvant activity, thereby enhancing the immuneresponse to an antigen (Nencioni et al., J. Immunol. 139:800-804 (1987);EP285441 to Howard et al.).

SUMMARY OF THE INVENTION

[0004] This invention pertains to vaccine compositions comprising amixture of one or more pneumococcal or meningococcal antigens, theinterleukin IL-12 and a mineral in suspension. The IL-12 can be eitheradsorbed onto the mineral suspension or simply mixed therewith. In aparticular embodiment of the invention, the IL-12 is adsorbed onto amineral suspension such as alum (e.g., aluminum hydroxide or aluminumphosphate). These vaccine compositions modulate the protective immuneresponse to the antigen; that is, the vaccine composition is capable ofquantitatively and qualitatively improving the vaccinated host'santibody response, and quantitatively increasing cell-mediated immunityfor a protective response to a pathogen. In a particular embodiment ofthe invention, the antigen is a pneumococcal or meningococcal antigen;the antigens are optionally conjugated to a carrier molecule, such as ina pneumococcal or meningococcal glycoconjugate.

[0005] Studies described herein show that IL-12 can modify the humoralresponse of mice immunized with pneumococcal and meningococcalglycoconjugate vaccines formulated with aluminum phosphate (AlPO₄) Theparticular pneumococcal polysaccharide serotypes exemplified herein areserotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F, and 23F, (Pn1, Pn4, Pn5, Pn6B,Pn9V, Pn14, Pn18C, Pn19F, Pn23F), and the meningococcal polysaccharideis type C (Men C). These serotypes, however, are not to be construed tolimit the scope of the invention, as other pneumococcal andmeningococcal serotypes are also suitable for use herein. Moreover, itwill be apparent to the skilled artisan that conjugation to a carriermolecule, such as the CRM₁₉₇ protein exemplified herein, is optional,depending upon the immunogenicity of the selected pneumococcal ormeningococcal antigen.

[0006] Doses of IL-12 ranging from about 8 ng to about 1,000 ngincreased the IgG1, IgG2a, IgG2b and IgG3 response to alum-adsorbed Pn14or Pn6B. In addition they increased the IgG2a response to Pn4 and Pn9V.Doses of IL-12 of about 5,000 ng markedly reduced the overall IgG titersto Pn14, and especially the IgG1 and IgG2b titers.

[0007] The invention also pertains to methods for preparing animmunogenic composition or a vaccine composition comprising a mixture ofantigen and IL-12 with a mineral in suspension. In particular, the IL-12is adsorbed onto the mineral suspension. The invention also pertains tomethods for eliciting or increasing a vaccinee's IFN-γ-producing T cellsand complement-fixing IgG antibodies for a protective immune response,comprising administering to a mammalian, e.g., human or primate, host aneffective amount of a vaccine composition comprising a mixture ofantigen, IL-12 and a mineral in suspension in a physiologicallyacceptable solution. In particular, the IL-12 is adsorbed onto themineral suspension.

DETAILED DESCRIPTION OF THE INVENTION

[0008] Work described herein reveals the ability of IL-12 to increasethe immune response to alum-based pneumococcal vaccines, particularlyserotype 14 and serotype 6B pneumococcal glycoconjugate vaccines, andmeningococcal vaccines, particularly type C, to increase the proportionof complement-fixing IgG2a and IgG2b antibodies. As described herein,PnPs-14-CRM₁₉₇ vaccine comprises a serotype 14 pneumococcalpolysaccharide conjugated to a non-toxic mutant of diphtheria toxoid(cross-reacting material) designated CRM197, and PnPs6B-CRM197 vaccinecomprises a serotype 6B pneumococcal polysaccharide conjugated toCRM₁₉₇. IL-12 was compared to MPL® (3-O-deacylated monophosphoryl lipidA; RIBI ImmunoChem Research, Inc., Hamitton, Mont.), which in mice is apotent adjuvant for pneumococcal vaccines. In a separate experimentconducted in Balb/c mice, the effect of IL-12 on the cytokine profile ofthe CRM-specific T cells induced by the exemplary conjugate vaccines onalum was examined.

[0009] IL-12 is produced by a variety of antigen-presenting cells,principally macrophages and monocytes. It is a critical element in theinduction of TH-1 cells from naive T cells. Production of IL-12 or theability to respond to it has been shown to be critical in thedevelopment of protective TH-1-like responses, for example, duringparasitic infections, most notably Leishmaniasis (Scott et al., U.S.Pat. No. 5,571,515). The effects of IL-12 are mediated by IFN-γ producedby NK cells and T helper cells. Interleukin-12 (IL-12), originallycalled natural killer cell stimulatory factor, is a heterodimericcytokine (Kobayashi et al., J. Exp. Med. 170:827 (1989)). The expressionand isolation of IL-12 protein in recombinant host cells is described inInternational Patent Application WO 90/05147, published May 17, 1990.

[0010] The studies described herein reveal the utility of IL-12 as anadjuvant in a pneumococcal or meningococcal vaccine, and particularly apneumococcal or meningococcal glycoconjugate vaccine. Accordingly, thisinvention pertains to vaccine compositions comprising a mixture of suchan antigen, IL-12 and a mineral in suspension. In a particularembodiment of the invention, the IL-12 is adsorbed onto a mineralsuspension such as alum (e.g., aluminum hydroxide or aluminumphosphate). These vaccine compositions modulate the protective immuneresponse to the antigen; that is, the vaccine composition is capable ofeliciting the vaccinated host's complement-fixing antibodies for aprotective response to the pathogen. In a particular embodiment of theinvention, the antigen is a pneumococcal antigen, particularly apneumococcal polysaccharide; the pneumococcal antigen is optionallyconjugated to a carrier molecule, such as in a pneumococcalglycoconjugate. The particular pneumococcal polysaccharide serotypesexemplified herein are serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F, and 23F;however, these serotypes are not to be construed to limit the scope ofthe invention, as other serotypes are also suitable for use herein.

[0011] In another embodiment of the invention, the antigen is ameningococcal antigen, particularly a meningococcal polysaccharide; themeningococcal antigen is optionally conjugated to a carrier molecule,such as in a meningococcal glycoconjugate. Type C Neisseria meningitidisis exemplified herein; however, this type is not to be construed tolimit the scope of the invention, as other types are also suitable foruse herein.

[0012] IL-12 can be obtained from several suitable sources. It can beproduced by recombinant DNA methodology; for example, the gene encodinghuman IL-12 has been cloned and expressed in host systems, permittingthe production of large quantities of pure human IL-12. Also useful inthe present invention are biologically active subunits or fragments ofIL-12. Commercial sources of recombinant human and murine IL-12 includeGenetics Institute, Inc. (Cambridge, Mass.).

[0013] The antigen of this invention, e.g., a pneumococcal ormeningococcal antigen or a pneumococcal or meningococcal glycoconjugate,can be used to elicit an immune response to an antigen in a mammalianhost. For example, the antigen can be a serotype 14 or 6B pneumococcalpolysaccharide or a portion thereof which retains the ability tostimulate an immune response. Additional suitable antigens includepolysaccharides from other encapsulated bacteria and conjugates thereof,secreted toxins and outer membrane proteins.

[0014] The method comprises administering to the mammal, e.g., human orprimate, an immunologically effective dose of a vaccine compositioncomprising a mixture of an antigen, such as a pneumococcal antigen or apneumococcal conjugate, and an adjuvant amount of IL-12 adsorbed onto amineral in suspension.

[0015] As used herein, an “immunologically effective” dose of thevaccine composition is a dose which is suitable to elicit an immuneresponse. The particular dosage of IL-12 and the antigen will dependupon the age, weight and medical condition of the mammal to be treated,as well as on the method of administration. Suitable doses will bereadily determined by the skilled artisan. The vaccine composition canbe optionally administered in a pharmaceutically or physiologicallyacceptable vehicle, such as physiological saline or ethanol polyols suchas glycerol or propylene glycol.

[0016] The vaccine composition may optionally comprise additionaladjuvants such as vegetable oils or emulsions thereof, surface activesubstances, e.g., hexadecylamin, octadecyl amino acid esters,octadecylamine, lysolecithin, dimethyl-dioctadecylammonium bromide,N,N-dicoctadecyl-N′-N′bis (2-hydroxyethyl-propane diamine),methoxyhexadecylglycerol, and pluronic polyols; polyamines, e.g., pyran,dextransulfate, poly IC, carbopol; peptides, e.g., muramyl dipeptide,dimethylglycine, tuftsin; immune stimulating complexes; oil emulsions;liposaccharides such as MPL® and mineral gels. The antigens of thisinvention can also be incorporated into liposomes, cochleates,biodegradable polymers such as poly-lactide, poly-glycolide andpoly-lactide-co-glycolides, or ISCOMS (immunostimulating complexes), andsupplementary active ingredients may also be employed. The antigens ofthe present invention can also be administered in combination withbacterial toxins and their attenuated derivatives. The antigens of thepresent invention can also be administered in combination with otherlymphokines, including, but not limited to, IL-2, IL-3, IL-15, IFN-γ andGM-CSF.

[0017] The vaccines can be administered to a human or animal by avariety of routes, including but not limited to parenteral, intradermal,transdermal (such as by the use of slow release polymers),intramuscular, intraperitoneal, intravenous, subcutaneous, oral andintranasal routes of administration. The amount of antigen employed insuch vaccines will vary depending upon the identity of the antigen.Adjustment and manipulation of established dosage ranges used withtraditional carrier antigens for adaptation to the present vaccine iswell within the ability of those skilled in the art. The vaccines of thepresent invention are intended for use in the treatment of both immatureand adult warm-blooded animals, and, in particular, humans. Typically,the IL-12 and the antigen will be co-administered; however, in someinstances the skilled artisan will appreciate that the IL-12 can beadministered close in time but prior to or after vaccination with theantigen.

[0018] The pneumococcal and meningococcal antigens of the presentinvention can be coupled to a carrier molecule in order to modulate orenhance the immune response. Suitable carrier proteins include bacterialtoxins rendered safe by chemical or genetic means for administration tomammals and immunologically effective as carriers. Examples includepertussis, diphtheria, and tetanus toxoids and non-toxic mutant proteins(cross-reacting materials (CRM)), such as the non-toxic variant ofdiphtheria toxoid, CRM₁₉₇. Fragments of the native toxins or toxoids,which contain at least one T-cell epitope, are also useful as carriersfor antigens, as are outer membrane protein complexes. Methods forpreparing conjugates of pneumococcal antigens and carrier molecules arewell known in the art and can be found, for example, in Dick and Burret,Contrib Microbiol Immunol. 10:48-114 (Cruse J M, Lewis R E Jr, eds;Basel, Krager (1989)) and U.S. Pat. No. 5,360,897 (Anderson et al.).

[0019] The adjuvant action of IL-12 has a number of importantimplications. The adjuvanticity of IL-12 can increase the concentrationof protective functional antibodies produced against the antigen in thevaccinated organism. The use of IL-12 as an adjuvant can enhance theability of antigens which are weakly antigenic or poorly immunogenic toelicit an immune response. It may also provide for safer vaccinationwhen the antigen is toxic at the concentration normally required foreffective immunization. By reducing the amount of antigen, the risk oftoxic reaction is reduced.

[0020] Typically, vaccination regimens call for the administration ofantigen over a period of weeks or months in order to stimulate a“protective” immune response. A protective immune response is an immuneresponse sufficient to protect the immunized organism from productiveinfection by a particular pathogen or pathogens to which the vaccine isdirected.

[0021] As shown in the Examples, in an alum-formulated vaccine,comprising IL-12 adsorbed onto AlPO₄ and a serotype 14 or serotype 6Bpneumococcal polysaccharide conjugated to CRM₁₉₇, which normally inducesa response dominated by IgG1, 0.2 μg of IL-12 substantially increasedthe IgG2a and IgG3 subclasses in both Balb/c and Swiss Webster mice, buthad little or no effect on IgG1. Enhancement of IgG2b to Pn14 was seenwith Swiss Webster mice; 0.2 82 g of IL-12 had the same effect as 25 μgof MPL® on the IgG subclass response to Pn14, suggesting that IL-12 isat least 100-fold more biologically active than MPL® in this regard. Asexpected from the IgG subclass distribution, especially the enhancedIgG2a response, the opsonophagocytic activity of the antisera for Pn14pneumococci from mice receiving 0.2 μg IL-12 was higher than that ofcontrols and was equivalent to that of mice immunized with vaccineformulated with a much larger amount of MPL®.

[0022] Briefly, IgG2a and IgG2b antibodies are very efficient atactivating the complement system, whereas IgG1 antibodies are not. Thecomplement system consists of a series of plasma proteins which cometogether around IgG2a or IgG2b bound to antigen (e.g., bacteria) to forma large molecular complex. Deposition of this complex on the surface ofbacteria results in the killing of the bacteria by perforating the cellmembrane (bactericidal activity) or by facilitating the recognition ofthe bacteria by phagocytic cells (such as polymorphonuclear cells (PMN)used in this study), which take up the bacteria and kill them(opsonophagocytosis).

[0023] Increasing the dose of IL-12 profoundly reduced the IgG1 andIgG2b responses. The reduction in these immunoglobulin subclasses wasnot simply due to a change in the kinetics of the antibody response, ashas been observed in the hen egg lysozyme (HEL) system (Buchanan, VanCleave and Metzger, Abstract #1945; 9th International Congress ofImmunology (1995)), as these subclasses were reduced at all time pointstested. The effect on IgG1 was expected given that switching of B cellsto this subclass requires IL-4, a TH-2 cytokine whose production isinhibited by IL-12. The reduction in IgG2b, however, was not expectedsince in previous studies increased levels of IgG2b have correlated withthe presence of TH-1-like T cells. It is likely that cytokines otherthan, or in addition to, IFN-γ are involved in regulation of IgG2b. Forexample, Germann et al. (Eur J. Immunol 25:823-829 (1995)) found thattreating mice with anti-IFN-γ inhibited the ability of IL-12 to promoteIgG2a responses, but not IgG2b. Other studies have implicated TGF-β asan important factor in the induction of IgG2b (reviewed by J. Stavnezer,J. Immunol. 155:1647-1651 (1995)). Without wishing to be bound bytheory, it is possible that high doses of IL-12 may affect TGF-βproduction or responsiveness to it. IFN-γ is critical for the inductionof IgG2a antibodies to T-dependent protein antigens (Finkelman andHolmes, Annu. Rev. Immunol. 8:303-33 (1990)) and IgG3 responses toT-independent antigens (Snapper et al., J. Exp. Med. 175:1367-1371(1992)). Increased IFN-γ response was consistently found after a singlevaccination with vaccine (PnPs-14-CRM₁₉₇) containing IL-12 and AlPO₄ andafter boosting. The effect of IL-12 on the TH-2 cytokines IL-5 and IL-10appears to depend on when the lymphoid cells are harvested aftervaccination, and possibly on the particular cytokine. Exogenous IL-12completely abolished antigen-specific IL-5 and IL-10 production by lymphnode cells (LNC) harvested 1 week after primary vaccination. Aftersecondary vaccination, differences were seen between these twocytokines; IL-5 production by either LNC or splenocytes was completelyabolished by 1 μg IL-12 in the vaccine, but IL-10 production was largelyunaffected after boosting. It is unclear whether these differences aredue to setting up the cultures at different times or reflect theexpansion of a TH-2-like population upon subsequent revaccination. Thelatter possibility is consistent with data from Wolf and colleagues(Bliss et al., J. Immunol 156:887-894 (1996)), indicating thatIL-4-producing T cells can be recovered from Balb/c mice previouslyimmunized with vaccine containing IL-12 and boosted with solubleantigen. In their studies, IL-4 was detected even if IL-12 was includedin the secondary vaccine. The presence of TH-2 cytokines after boostingmay explain why, in Balb/c mice, even high levels of IL-12 could notreduce the secondary IgG1 response to below control levels (conjugatevaccine on alum). Unlike the Balb/c mice, high doses of IL-12 severelyinhibited the IgG1 response of Swiss Webster mice. Whether this isassociated with decreased production of TH-2 cytokines after the secondvaccination is unclear.

[0024] In the present studies, IL-12 exhibited either onlyimmunomodulatory activity or behaved both as a “classical” adjuvant, anda immunomodulator, depending on the vaccine. In the study withPnPs14-CRM₁₉₇ the IgG response (especially the primary response) to thevaccine was not substantially elevated by the presence of the cytokinebut certain subclasses, i.e. IgG2a and IgG3, were elevated whereas theothers were unchanged or diminished. Thus, IL-12 is useful for alteringthe humoral response to an already immunogenic vaccine. It is possiblethat in these studies the adjuvant activity of IL-12 was masked by thepresence of alum, which is an adequate adjuvant on its own for thehighly immunogenic PnPs-14 conjugate. The adjuvanticity of IL-12 may bebetter demonstrated in the absence of alum, by reducing the dose ofconjugate or by using a poorly immunogenic conjugate. Thus, furtherevaluations were carried out using IL-12 in the presence and absence ofalum with PnPs6B conjugate vaccines, which are less immunogenic in SwissWebster mice than PnPs-14 conjugate vaccines.

[0025] An additional study was designed to address the issue of IL-12adjuvant activity for a poorly immunogenic pneumococcal conjugate. ThePn18C conjugate was chosen, as it is poorly immunogenic when formulatedwith AlPO₄, i.e., it induces low IgG titers and not all mice respond toit. When formulated with MPL or QS-21, higher IgG titers and a greaterfrequency of responders can be achieved.

[0026] One hundred μg MPL® plus AlPO₄ or 20 μg QS-21™ were the bestadjuvants in this study for a Pn18C response as they induced the highestfrequency of responders to this serotype. Nonetheless, IL-12 had markedeffects on the IgG response to the carrier protein, CRM₁₉₇, in miceimmunized with this conjugate. Moreover, the effects of the cytokinewere modified by the presence of AlPO₄ in the vaccine. IL-.12 clearlyacted as an adjuvant for vaccines formulated without AlPO₄, causing adose-dependent increase in IgG titers after primary and secondaryvaccination. IL-12 enhanced the IgG2a response to CRM₁₉₇, which isconsistent with its ability to favor the induction of TH-1-like helpercells (IFN-γ producers). However, IL-12 also enhanced the IgG1 responseto CRM₁₉₇ after primary and secondary vaccination. IgG1 antibodies arenormally associated with TH-2-like helper cells which produce IL-4.

[0027] Inclusion of 0.1 μg IL-12 into an AlPO₄-based Pn18C conjugatevaccine (which on its own induced a 10-fold higher CRM₁₉₇ response) hadno effect on IgG1 but substantially increased the IgG2a titer. The IgG2atiter achieved with 0.1 μg IL-12 was at least as high as that obtainedwith 5 μg IL-12 in the absence of AlPO₄. It should be noted, however,that the presence of AlPO₄ does not preclude the enhancement of IgG1responses by IL-12. In mice immunized with the Pn14 conjugate on AlPO₄,a 0.2 μg dose of IL-12 enhanced the IgG1, IgG2a and IgG2b titers toCRM₁₉₇. The differences in the effect on IgG1 may reflect differences inthe immunogenicity of the two conjugates for CRM₁₉₇ IgG responses; thePn14 conjugate on AlPO₄ induced 10-fold lower CRM₁₉₇ IgG titers so thatthere was room for IL-12 to enhance an IgG1 response, but not when micewere immunized with Pn18C conjugate on AlPO₄. The fact that MPL® andQS-21™ markedly increased the IgG1 titers in mice immunized with Pn18Cconjugate on AlPO₄ indicates that the IgG1 response had not beenmaximally stimulated. Alternatively, the nature of the saccharides onthe conjugates may be a factor. In both experiments, higher doses ofIL-12 resulted in a marked diminution of the IgG1, IgG2a and IgG2btiters to CRM₁₉₇, an effect that was not seen in the absence of AlPO₄.

[0028] IL-12 probably exerts its adjuvant effect differently than MPL®or QS₂₁™. IL-12 markedly enhanced the CRM₁₉₇ IgG2a titers in miceimmunized with Pn18C conjugate but had minimal effects on IgG2b. Incontrast, MPL® and QS-21™ enhanced the titers of both IgG subclasses.The dissociation of these two subclasses suggests that IgG2b is inducedby cytokines other than, or in addition to, the IFN-γ that drivesswitching to IgG2a and is known to mediate the immunomodulatory effectsof IL-12. One candidate for driving IgG2b production is TGFb. The natureof the antigen cannot be excluded, however, since in mice immunized withPn14 conjugate, 0.2 μg IL-12 caused IgG2a and IgG2b to be elevated tosimilar levels which were equivalent to the titers promoted by 25 μgMPL®.

[0029] Studies utilizing a bivalent vaccine consisting of aPnPs14-CRM₁₉₇ conjugate mixed with a conjugate of capsularpolysaccharide from serotype 6B pneumococci covalently linked to CRM₁₉₇(PnPs6B-CRM₁₉₇) confirmed and extended the above-described findings.IL-12 not only modified the IgG response to the Pn6B conjugate, but alsoenhanced the overall IgG titer to the conjugate. Moreover, this workfurther demonstrates that the adjuvant activity of relatively low dosesof IL-12 is enhanced by formulating it with AlPO₄. Unlike theabove-described studies with PnPs-14-CRM₁₉₇ glycoconjugate, IL-12/AlPO₄enhanced both the IgG1 and IgG2a subclasses to Pn6B, indicating that theapparent lack of enhancement of the Pn14 IgG1 response by IL-12 isprobably not a generalizable phenomenon. This work further supports theidea that the mechanisms of adjuvant activity by IL-12 and MPL® are notequivalent. Both adjuvants enhanced the Pn6B IgG1 and IgG2a titers tosimilar levels, but MPL® was more effective at promoting IgG2b and IgG3antibodies.

[0030] IL-12/AlPO₄ did not act as an adjuvant for the Pn14 IgG response.The reason for this is not clear; however, without wishing to be boundby theory, this most likely reflects the fact that in previous studiesmice were immunized with a 1 μg dose of PnPs-14-CRM₁₉₇ glycoconjugate,i.e., 10-fold higher than in the Pn6B studies. The applicability ofIL-12 to more complex pneumococcal vaccines was demonstrated using anonavalent vaccine containing glycoconjugates from serotype 1, 4, 5, 6B,9V, 14, 18C, 19F and 23F pneumococci. The combination of IL-12 withAlPO₄ enhanced the IgG2a antibodies to PnPs4 and PnPs9V, in addition toPnPs6B and PnPs14, and increased the ability of mice to respond toglycoconjugate prepared with serotype 18C pneumococcal saccharide(PnOs-18C-CRM₁₉₇) which is poorly immunogenic in mice.

[0031] In further examples, IL-12 was tested with a glycoconjugatevaccine against type C Neiserria meningitidis (MenC) and aglycoconjugate vaccine against type B Hemophilus influenzae (HbOC).Formulating that vaccine with 50 ng IL-12 and AlPO₄ enhanced the IgG2atiters to MenC capsular polysaccharide although not to HbOC.

[0032] The data presented herein indicate that AlPO₄ can greatly enhancethe potency of IL-12 so that substantially lower doses of the cytokinecan be used. One possible mechanism is that IL-12 binds to AlPO₄,thereby enhancing its persistence in the animal; additional studiesindicate that IL-12 rapidly binds to alum (data not shown).Alternatively, the local inflammatory effect of AlPO₄ may inducecytokines that potentiate the biological activity of IL-12.

[0033] In addition to understanding the physical interaction of IL-12with AlPO₄, several other issues arise from the present work withpneumococcal vaccines formulated with IL-12. Given that AlPO₄ enhancesthe activity of IL-12, it would be useful to know the minimal dose ofcytokine needed to adjuvant the IgG response to pneumococcalglycoconjugates, as well as whether IL-5-producing T cells are activatedby IL-12-containing glycoconjugate vaccines. These two questions wereaddressed in the studies in Balb/c mice described in Example 4.

[0034] The following Examples are offered for the purpose ofillustrating the present invention and are not to be construed to limitthe scope of this invention. The teachings of all references citedherein are hereby incorporated herein by reference.

EXAMPLES Example 1

[0035] Effect of IL-12 on the IgG Response of Swiss Webster Mice toSerotype 14 Pneumococcal Capsular Polysaccharide Conjugated to CRM₁₉₇ onAluminum Phosphate (PnPs-14-CRM/AlPO₄)

[0036] Study Design

[0037] Swiss Webster mice (10 per group) were immunized twice (at weeks0 and 3) with 1 μg PnPs-14-CRM₁₉₇ formulated with 100 μg AlPO₄ andeither no IL-12, 0.2 μg, 1 μg or 5 μg IL-12. All vaccines included 0.25%normal mouse serum for the purpose of stabilizing the IL-12 when used atlow concentrations. PnPs14-CRM₁₉₇ is a conjugate of capsularpolysaccharide from serotype 14 pneumococci covalently linked to thegenetically detoxified diphtheria toxin, CRM₁₉₇, by reductive amination.Another group received 25 μg MPL® (3-O-deacylated monophosphoryl lipidA, RIBI Immunochem Research, Inc., Hamilton Mont.) instead of IL-12. Thevaccinations were given subcutaneously three weeks apart. Sera werecollected at week 3 (primary response) and weeks 5 and 7 (secondaryresponses 2 and 4 weeks after boosting). The sera were analyzed for IgGantibodies to PnPs-14.

[0038] The sera were also analyzed for the ability to promoteopsonophagocytic killing of type-14 pneumococci by humanpolymorphonuclear cells (PMN). Type 14 pneumococci were opsonized withdilutions of antisera and C8-depleted serum as a source of complement.They were then incubated with human polymorphonuclear cells (PMN), andthe percent of bacteria surviving was determined by colony counts.

[0039] Results

[0040] Table 1 shows that 1 μg and 5 μg IL-12 substantially reduced theanti-PnPs-14 IgG response in mice immunized with conjugate formulatedwith AlPO₄. The lowest dose (0.2 μg) of cytokine had no effect on thetotal IgG response but caused major changes in the levels of theindividual immunoglobulin subclasses. At weeks 5 and 7 (2 and 4 weeksafter boosting, respectively), 0.2 μg IL-12 induced substantially higherIgG2a, IgG2b and IgG3 titers but left the IgG1 levels essentiallyunaltered. The IgG subclass profile induced by 0.2 μg IL-12 wasindistinguishable from that obtained with 25 μg MPL®, and sera from micereceiving these adjuvants had higher opsonophagocytic activity thanthose from mice immunized with a vaccine containing only AlPO₄ (Table2).

[0041] The higher doses of IL-12 markedly reduced the IgG1 antibodies;at 5 μg cytokine, IgG1 titers were at least 10-fold lower than in miceimmunized without IL-12. This effect was apparent both during theprimary response and after boosting. Increasing the IL-12 dose did notcause further increases in IgG2a, IgG2b and IgG3, and, like IgG1, theyalso declined, although to varying degrees. IgG2b showed the greatestreduction such that vaccines containing 1 μg or 5 μg IL-12 induced thesame IgG2b titer as those without adjuvant. IgG2a and IgG3 were lesssensitive to the effects of high IL-12 dose; even with 5 μg IL-12, afterthe second vaccination these subclasses were higher than in thecontrols.

[0042] These studies showed that IL-12 could modulate the IgG subclassresponse to a PnPs14-CRM₁₉₇ conjugate vaccine formulated with AlPO₄. A0.2 μg dose of IL-12 increased the IgG2a, IgG2b and IgG3 response toPn14 without affecting the IgG1 response. Higher doses of IL-12 resultedin a marked reduction in the IgG1 and IgG2b titers. IgG2a and IgG3titers also appeared to decline at these doses, but they were stillhigher than in mice immunized in the absence of IL-12. Example 2demonstrates that the IgG subclass changes were associated with enhancedinduction of IFN-γ-producing, CRM₁₉₇-specific T cells and a markedreduction in antigen-specific IL-5 production, suggesting a change inthe T helper cell phenotype from TH-2-like to TH-1-like. TABLE 1 Effectof IL-12 on the immunogenicity of PnPs-14- CRM₁₉₇/alum vaccine Adju-Dose PnPs14 IgG Response Time vant (μg) IgG IgG1 IgG2a IgG2b IgG3 week 3None 56,035 8,394 481 298 1,312 IL-12 5 13,137 480 2,417 388 2,398 IL-121 26,131 1,521 3,249 736 3,858 IL-12 0.2 90,220 13,779 4,731 1,454 7,944MPL ® 25 46,451 14,303 1,506 8,506 18,203 week 5 None 531,270 189,5715,507 14,463 18,158 IL-12 5 231,015 16,900 28,719 6,002 56,982 IL-12 1198,044 36,327 27,420 11,841 30,740 IL-12 0.2 722,360 305,623 60,70189,397 99,794 MPL ® 25 751,066 221,324 44,957 91,265 77,989 week 7 None694,741 244,212 1,801 6,849 9,245 IL-12 5 177,438 17,232 20,276 3,49426,859 IL-12 1 183,571 44,213 21,246 5,063 13,447 IL-12 0.2 852,292251,157 37,104 37,717 88,933 MPL ® 25 783,622 187,055 30,694 89,15359,297

[0043] TABLE 2 Opsonophagocytic activity of sera of mice immunized withPnPs-14-CRM₁₉₇/AlPO₄ formulated with IL-12 or MPL ® Initial % BacteriaSurviving Serum Week 5 Sera Week 7 Sera Dilution No 0.2 μg 1 μg 5 μg 25μg No 0.2 μg 1 μg 5 μg 25 μg Tested IL-12 IL-12 IL-12 IL-12 MPL ® IL-12IL-12 IL-12 IL-12 MPL ® 2 6 10 6 6 9 6 6 7 7 5 8 12 7 9 9 7 21 4 10 9 616 32 4 24 25 3 47 8 17 26 8 32 71 12 61 94 23 68 6 85 90 21 64 64 46 9089 51 116 34 79 99 76

Example 2

[0044] Nature of T Helper Cells Induced by Pneumococcal ConjugateVaccine (PnPs-14-CRM₁₉₇/AlPO₄) Formulated with IL-12

[0045] Study Design

[0046] Groups of eight (8) Balb/c mice were immunized subcutaneously atthe base of the tail with 1 μg PnPs-14-CRM₁₉₇ conjugate formulated with100 μg AlPO₄ and different doses of IL-12. Normal mouse serum (0.25%)was included as a carrier protein. One week later, draining lymph nodecell suspensions were prepared from half the mice in each group andcultured with CRM₁₉₇, lysozyme, ConA or in medium alone for 6 days.Culture supernatants from parallel cultures were harvested at day 3 andday 6 and assayed for IFN-γ, IL-5 and IL-10 by ELISA.

[0047] At three weeks, the remaining mice were bled and reimmunized withthe same vaccine formulation used in the first immunization. Fourteendays after the second immunization (week 5), the mice were bled oncemore. Four days later their draining lymph node cells and splenocyteswere harvested and cultured for six days with CRM₁₉₇, lysozyme, ConA orin medium alone. Culture supernatants from parallel cultures wereharvested at day 3 and day 6 and assayed for IFN-γ, IL-5 and IL-10 byELISA.

[0048] Results

[0049] Formulating PnPs-14-CRM₁₉₇/AlPO₄ vaccine with the lower doses ofIL-12 (0.2 μg and 1.0 μg) greatly enhanced the IgG2a and IgG3 responsesto Pn14 at week 5, but not IgG1 (see Table 3). Several differences wereseen between the results obtained with Balb/c mice and Swiss Webstermice in the previous experiment; in this experiment IL-12 did notdramatically increase the IgG2b antibodies to Pn14, nor did the 5 μgIL-12 dose cause the dramatic (>10-fold) reduction in IgG1 titersrelative to the control group without cytokine.

[0050] One week after immunization, lymph node cells from mice immunizedwithout IL-12 produced IFN-γ, IL-5 and IL-10 when stimulated with CRM₁₉₇in vitro (Table 4) Adding IL-12 to the vaccine dramatically increasedthe antigen-specific production of IFN-γ and abolished the ability ofthe lymphoid cells to produce IL-5 and IL-10. Maximal IFN-γ productionwas obtained with the lowest dose of IL-12 (0.2 μg); higher doses,particularly 5 μg, appeared to reduce the levels of this cytokine. Thiswas most clearly seen in cultures stimulated with 1 μg/mL CRM₁₉₇. Thereduction in IFN-γ with higher doses of IL-12 may not reflect ageneralized suppressive phenomenon since IFN-γ production in response toCon A was the same regardless of the dose of IL-12 in the vaccine.

[0051] Two weeks after the second immunization, lymph node cells andsplenocytes from mice immunized with vaccine containing IL-12 continuedto produce elevated levels of IFN-γ in response to stimulation withCRM₁₉₇ compared to mice immunized without IL-12 (Table 5). As observed 7days after primary vaccination, 0.2 μg to 1.0 μg IL-12 were optimaldoses of IL-12 for augmentation of an IFN-γ response. In contrast,however, IL-5 and IL-10 production were differentially affected. The 1.0and 5.0 μg doses of IL-12 essentially eliminated the IL-5 response but,by comparison, had only a-minor effect on IL-10 production. IL-12 (5.0μg) abolished the ability of splenocytes but not lymph node cells toproduce IL-10 (Tables 5 and 6). TABLE 3 Effect of IL-12 on immuneresponse to an alum- based PnPs14 glycoconjugate vaccine in Balb/c MiceWeek IL-12 Dose IgG IgG1 IgG2a IgG2b IgG3 3 none 41,480 7,347 1,387 8952,333 0.2 26,253 1,521 1,118 171 5,911 1 26,124 966 2,155 248 5,991 510,753 541 671 183 3,242 5 none 234,220 33,284 2,896 3,105 2,487 0.2674,996 71,808 18,245 6,789 107,470 1 632,714 32,022 22,749 7,853 44,3505 224,832 19,495 10,083 1,287 25,212

[0052] TABLE 4 Cytokines produced by lymph node cells taken 7 days aftersingle immunization with PnPs-14 conjugate formulated with AlPO₄ andIL-12 Day 6 Cultures Antigen No 0.2 μg 1.0 μg 5.0 μg Cytokine in vitroμg/ml IL-12 IL-12 IL-12 IL-12 IFN-γ CRM 30 23.2 102.7 60.5 32.2 (U/mL)CRM 1 <0.75 65.2 28.6 8.7 Lysozyme 30 <0.75 2.9 6.6 4.5 Con A 1 43.897.1 107.1 105.4 Medium — <0.75 3.6 10.6 5.2 IL-5 CRM 30 7.2 <0.22 <0.22<0.22 (ng/mL) CRM 1 2.2 <0.22 <0.22 <0.22 Lysozyme 30 <0.22 <0.22 <0.22<0.22 Con A 1 <0.22 <0.22 <0.22 <0.22 Medium — <0.22 <0.22 <0.22 <0.22IL-10 CRM 30 10.4 0.8 0.21 0.21 (ng/mL) CRM 1 2.6 0.21 0.21 0.21Lysozyme 30 <0.14 0.21 0.21 0.21 Con A 1 <0.14 0.21 0.21 0.21 Medium —<0.14 0.21 0.21 0.21

[0053] TABLE 5 Cytokine production by splenocytes two weeks aftersecondary vaccination with PnPs-14 conjugate formulated with AlPO₄ andIL-12 Day 6 Cultures Antigen No 0.2 μg 1.0 μg 5.0 μg Cytokine in vitroμg/ml IL-12 IL-12 IL-12 IL-12 IFN-γ CRM 30 7.0 98.4 83.2 50.9 (U/mL) CRM1 1.0 89.2 76.8 16.4 Lysozyme 30 <0.4 <0.4 <0.3 <0.3 Con A 1 42.7 48.750.6 49.5 IL-5 CRM 30 13.2 3.1 0.6 <0.2 (ng/mL) CRM 1 4.5 4.4 0.8 <0.2Lysozyme 30 <0.3 <0.3 <0.2 <0.2 Con A 1 <0.3 <0.3 <0.2 <0.2 IL-10 CRM 308.6 4 7.1 0.6 (ng/mL) CRM 1 1.1 2.5 1.7 <0.2 Lysozyme 30 <0.2 <0.2 <0.3<0.2 Con A 1 0.5 0.4 <0.3 <0.2

[0054] TABLE 6 Cytokine production by lymph node cells two weeks aftersecondary vaccination with PnPs-14 conjugate formulated with AlPO₄ andIL-12 Day 6 Cultures Antigen No 0.2 μg 1.0 μg 5.0 μg Cytokine in vitroμg/ml IL-12 IL-12 IL-12 IL-12 IFN-γ CRM 30 9.8 86.9 58.7 62.0 (U/mL) CRM1 0.6 78.6 62.9 36.8 Lysozyme 30 <0.4 <0.4 <0.3 <0.3 Con A 1 17.7 57.645.7 69.0 IL-5 CRM 30 12.5 1.4 <0.2 0.5 (ng/mL) CRM 1 4.8 2.2 <0.2 <0.2Lysozyme 30 <0.3 <0.3 <0.2 <0.2 Con A 1 1.1 <0.3 <0.2 <0.2 IL-10 CRM 3011.3 9.9 7.2 3.6 (ng/mL) CRM 1 4.4 5.5 3.3 1 Lysozyme 30 <0.2 <0.2 <0.2<0.2 Con A 1 <0.2 <0.2 <0.2 <0.2

Example 3

[0055] IL-12 Adjuvant Activity with Poorly Immunogenic PneumococcalConjugate

[0056] Study Design

[0057] Swiss-Webster mice (10 per group) were immunized with 1 μg Pn18Cconjugate formulated with or without 100 μg ALPO₄. The vaccines weresupplemented with either IL-12 (0.2, 1 or 5 μg), 100 μg MPL® or 20 μgQS-21™. Normal mouse serum (0.5% final) was used to stabilize thediluted IL-12 and was added to all vaccines, regardless of composition.Three weeks later, the mice were bled and boosted with the same vaccineformulation used at the primary immunization. Bleeds were also taken atweeks 5 and 7 of the study (2 and 4 weeks after boosting, respectively).Pooled sera were tested at week 5 for Pn18C and CRM₁₉₇ total IgG and IgGsubclasses. To determine the frequency of responders to Pn18C, the serafor individual mice were diluted 1/500 and tested by ELISA for IgGantibodies to Pn18C. Results are reported as optical density.

[0058] Results

[0059] The Pn18C IgG responses are presented in Table 7. The addition ifIL-12 to alum-formulated conjugate vaccine had no consistent effect onthe IgG response to Pn18C. A dose of 5 μg of IL-12 caused a 3-fold risein the IgG titer of pooled week 5 sera, whereas vaccine formulated with1 μg of IL-12 appeared to induce no Pn18C response. The lowest dose ofIL-12 (0.1 μg) induced the same response as the AlPO₄-formulated vaccinenot containing IL-12. The vaccine formulated with MPL®/AlPO₄ induced thehighest frequency of responses; 7/10 mice gave OD>0.2, in contrast toQS-21™/AlPO₄ and AlPO₄ alone, each of which induced 4/10 responders.Mice immunized with vaccine containing IL-12 plus AlPO₄ induced 2/10,0/10 and 1/10 responders at IL-12 doses of 0.1 μg, 1.0 μg, and 5 μg,respectively.

[0060] In this experiment MPL® and QS-21™ caused at most a 3- to 4-foldincrease in the Pn18C IgG response. In the absence of AlPO₄, IL-12 didnot have a profound adjuvant effect on the Pn18C IgG response. Thevaccine containing a 1 μg dose of IL-12 induced the same Pn18C responseas vaccine without IL-12. Vaccines containing the lower and higher dosesof IL-12 appeared to induce lower responses than the control vaccine.Neither MPL® nor QS-21™ appeared to enhance the Pn18C IgG response.Among the vaccines formulated without AlPO₄, QS-21™ induced the highestfrequency of responders (7/10 with OD>0.2), whereas all otherformulations induced 4/10 responders, at most.

[0061] To confirm that the IL-12 in the vaccine was indeed active, theCRM₁₉₇ IgG response in these mice was evaluated. Tables 8 and 9 showthat after primary (week 3) and secondary (week 5) vaccination, IL-12causes a dose-dependent increase in CRM₁₉₇ IgG response in miceimmunized with vaccine formulated without AlPO₄. Moreover, there was anIL-12 dose-dependent increase in both IgG1 and IgG2a titers at weeks 3and 5, as well as an increase in IgG2b at week 5. The IgG1 and IgG2atiters at week 5 were similar to those induced by vaccine formulatedwith 100 μg MPL®. In contrast, the IgG2b titers promoted by IL-12 were20-fold lower than those induced by MPL®. These data suggest that IgG2aand IgG2b are controlled by different mechanisms, IgG2a being dependenton a mechanism activated by IL-12 and IgG2b being controlled by anIL-12-independent mechanism. These data clearly indicate that IL-12 canact as adjuvant for IgG responses to a protein antigen. Moreover theincrease in both IgG1 and IgG2a titers suggest that, within this modelat least, IL-12 enhances priming of both TH-1-like and TH-2-like helpercells by PnOs18C-CRM₁₉₇ conjugate in the absence of AlPO₄.

[0062] When added to the Pn18C conjugate vaccine formulated with AlPO₄,the 0.1 μg dose of IL-12 caused little if any increase in the week 3total IgG response to CRM₁₉₇ but a 3-fold increase at week 5. However,this dose of IL-12 increased the IgG2a titer at week 5, promoting titerssimilar to that induced by vaccine containing MPL or QS-21. IL-12 didnot markedly increase the IgG2b titers. As seen in previous experiments,higher doses of IL-12 resulted in a sharp decline in IgG titers with allsubclasses being affected. TABLE 7 Effect of IL-12 on IgG response toPnOs18C conjugate IgG titer IgG subclasses at Week 5 Adjuvant (μg/dose)Wk3 Wk5 IgG1 IgG2a IgG2b IgG3 AlPO₂ <100 4,608 4,591 116 <100 <100 0.1μg IL-12 + <100 3,681 1,472 265 259 450 AlPO₄ 1.0 μg IL-12 + <100 130<100 <100 <100 <100 AlPO₄ 5.0 μg IL-12 + 260 13,545 7,820 1,426 <1001,481 AlPO₄ 100 μg MPL/AlPO₄ 233 9,027 1,522 935 877 <100 QS-21 + AlPO₄<100 7,989 879 1,395 1,062 1,004 none 107 10,768 5,238 345 <100 144 0.1μg IL-12 <100 1,808 336 105 <100 <100 1.0 μg IL-12 <100 22,257 12,443671 172 773 5.0 μg IL-12 <100 460 203 <100 <100 400 100 μg MPL 112 1,729524 363 189 126 QS-21 <100 3,573 2,483 101 <100 113

[0063] TABLE 8 Effect of IL-12 on CRM₁₉₇ IgG response three weeks aftervaccination with PnOs18C conjugate Adjuvant (μg/dose) IgG IgG1 IgG2aAlPO₄ 70,964 8,706 3,516  0.1 μg IL-12 + AlPO₄ 103,589 4,754 13,025  1.0μg IL-12 + AlPO₄ 26,927 506 2,926  5.0 μg IL-12 + AlPO₄ 19,579 241 2,665100 μg MPL ^(®)/AlPO₄ 651,315 92,245 79,508 QS-21 ™/ALPO₄ 572,255116,583 38,419 None 7,630 452 1,023  0.1 μg IL-12 32,403 3,475 3,713 1.0 μg IL-12 60,987 4,615 5,951  5.0 μg IL-12 128,697 10,498 10,686 100μg MPL ^(®)/TEM 462,289 40,010 24,979 QS-21 ™ 556,440 111,533 53,799

[0064] TABLE 9 Effect of IL-12 on CRM197 IgG response five weeks aftervaccination with PnOs18C conjugate (two weeks after boosting) Adjuvant(μg/dose) IgG IgG1 IgG2a IgG2b AlPO₄ 634,631 102,974 45,955 8,812  0.1μg IL-12 + AlPO₄ 2,225,000 88,204 317,083 16,869  1.0 μg IL-12 + AlPO₄105,765 8,018 12,598 1,096  5.0 μg IL-12 + AlPO₄ 71,618 1,582 13,806 744100 μg MPL ^(®)/AlPO₄ 4,384,000 637,655 371,652 111,646QS-21 ™/ALPO₄ >5,000,000 >1,000,000 873,674 144,132 None 62,341 12,7833,655 1,679  0.1 μg IL-12 296,791 52,288 23,741 7,069  1.0 μg IL-121,026,060 101,381 96,024 11,862  5.0 μg IL-12 1,367,771 74,494 108,81514,258 100 μg MPL ^(®)/TEM 4,173,765 264,691 266,160 303,662QS-21 ™ >5,000,000 1,303,508 445,712 131,991

Example 4

[0065] Effect of IL-12 on the IgG Response of Swiss Webster Mice toBivalent Vaccine Containing PnPs6B-CRM₁₉₇ and PnPs-14-CRM₁₉₇

[0066] Study Design

[0067] Swiss Webster mice were immunized subcutaneously at weeks 0 and 3with a vaccine comprising 0.1 μg per dose of PnPs6B-CRM₁₉₇glycoconjugate (a conjugate of capsular polysaccharide from serotype 6Bpneumococci covalently linked to CRM₁₉₇) plus 0.1 μg per dose ofPnPs14-CRM₁₉₇ glycoconjugate. The vaccines were administered with 0, 8,40, or 200 ng IL-12, either alone or in combination with 100 μg alum(AlPO₄). Normal mouse serum (0.25%) was included as a carrier protein tostabilize the IL-12 at low concentrations. A control group of mice wasimmunized with the vaccine formulated with 100 μg monophosphoryl lipid A(MPL®). The mice were bled at week 3 (primary response) and week 5(secondary response). Sera were tested for IgG antibodies to Pn6B andPn14 capsular polysaccharide by ELISA.

[0068] Results

[0069] Response to PnPs6B Conjugate

[0070] Table 10 illustrates the pooled serum IgG response to the Pn6Bcomponent of the bivalent vaccine. Little or no response to Pn6B wasdetected at week 3 if the vaccine contained no adjuvant or wasformulated with only AlPO₄. The highest titers after a singlevaccination appeared to be induced by vaccine containing either, MPL® or8-40 ng of IL-12 co-formulated with alum. These titers however were low,i.e., less than 3,000. The week 5 responses show that after boosting,vaccines formulated with 40 ng IL-12 plus AlPO₄ or with MPL® induced thehighest IgG titers to Pn6B. In the absence of alum, IL-12 in the 8 to200 ng dose range did not enhance the IgG titers to Pn6B.

[0071] The IgG subclass response to Pn6B at week 5 is shown in Table 10.The titers of the individual IgG subclasses were similar in miceimmunized with vaccine containing no adjuvant or vaccine formulated withAlPO₄ (no IL-12). Moreover, formulating the vaccines with 8-200 ng ofIL-12 in the absence of AlPO₄ did not alter the IgG subclass response.In contrast, these doses of IL-12 when combined with AlPO₄ resulted insubstantially increased IgG1 and IgG2a titers to Pn6B. These titers weresimilar to those obtained with vaccine formulated with MPL®. IL-12 alsoincreased the IgG2b and IgG3 titers induced by vaccine formulated withAlPO₄; however, these titers appeared to be substantially lower thanthose induced by vaccine formulated with MPL®.

[0072] To determine if the increases obtained with a combination ofIL-12 and AlPO₄ were statistically significant, the Pn6B IgG titers ofindividual mice in selected groups were determined. The geometric meantiters (GMT) are presented in Table 11. The data indicate that groupsimmunized with vaccines formulated without adjuvant or with AlPO₄ alonehad similar GMT against Pn6B. Formulating the vaccine with AlPO₄ plus 40ng IL-12 resulted in a 29-fold increase in titer over that induced byvaccine containing no adjuvant. When all the data were tested by ANOVA(analysis of variance by JMP software; SAS Institute, Cary, N.C.), nostatistically significant differences were found. Upon comparison ofsubsets of data, ANOVA indicated a statistically significant differencewhen comparing the week 5 responses induced by vaccine containing noadjuvant and vaccines formulated with AlPO₄ and various doses of IL-12.Of these, the vaccine formulated with AlPO₄ plus 40 ng IL-12 induced asignificantly higher Pn6B titer than vaccine formulated withoutadjuvant. As a further indication of the heightened immunogenicity ofthat formulation, 7 of the 10 mice in that group had Pn6B titers greaterthan or equal to 50,000 compared to only 1 and 2 mice each in the groupsvaccinated with conjugate formulated without adjuvant or with AlPO₄alone. TABLE 10 Effect of IL-12 on the IgG response to PnPs6B in miceimmunized with a bivalent PnPs6B/14 pneumococcal glycoconjugate vaccinePn6B IgG Subclass Response at Pn6B IgG Titer* Week 5* Group AdjuvantWeek 3 Week 5 IgG1 IgG2a IgG2b IgG3 P344 200 ng IL-12 + 630 161,86719,474 22,664 2,954 7,333 P345 40 ng IL-12 + 2,609 429,006 61,364 24,1724,117 9,830 P346 8 ng IL-12 + AlPO₄ 1,977 284,206 46,734 32,859 3,1958,764 P347 AlPO₄ (no IL-12) 279 120,999 8,767 2,199 688 301 P348 200 ngIL-12 <100 22,401 6,816 3,147 501 1,104 P349 40 ng IL-12 164 23,3435,056 2,532 879 292 P350 8 ng IL-12 642 81,748 17,702 3,573 5,151 1,786P351 None <100 20,153 3,061 1,506 364 1,220 P352 100 μg MPL ^(®) 2,872840,513 84,660 30,813 43,505 25,749

[0073] TABLE 11 Pn6B IgG titers of individual mice P344 P345 P346 P347P350 P351 P352 Group AlPO₄ + 200 ng AlPO₄ + 40 ng AlPO₄ + 8 ng AlPO₄ 8ng No 100 μg Mouse # IL-12 IL-12 IL-12 (no IL-12) IL-12 Adjuvant MPL ® 11,957 596,886 13,457 306,012 833,148 3,544 7,556 2 2,498 1,205 1,000,0003,653 9,431 326 1,359,470 3 100 9,453 1,422 8,708 3,163 1,136 81 411,830 70,278 168,481 41,395 109,399 24,140 583,097 5 1,823 157,42716,454 677,407 252 50,785 86,656 6 6,114 90,843 989 9,089 150,245 228284 7 279 49,182 372,709 17,164 112 1,351 1,000,000 8 756,503 408,348425 7,329 393 36,805 473,652 9 1,000,000 1,000,000 667,988 100 13,62222,817 927,213 10 177 1,052,210 6,206 245 182,629 851 — GMT 4,347103,743 22,580 9,735 10,120 3,589 55,799 Mice with 2 7 4 2 4 1 6 titer ≧50,000

[0074] Response to PnPs14 Conjugate

[0075] The IgG response to the PnPs14 component of the vaccine is shownin Table 12. The data indicate that IL-12 in the 8-40 ng dose range,either alone or when formulated with AlPO₄, did not enhance the responseto PnPs14 after primary or secondary vaccination. Moreover, subclassanalysis indicated that IL-12 did not enhance the IgG2a titers whenformulated with IL-12. In this study, MPL® did not have the profoundadjuvant effect on the PnPs14 response that was observed in previousstudies, at least when assaying pooled sera. To get an idea of thedegree of variation of the response of each group, individual sera wereassayed for Pn14 IgG antibodies at a {fraction (1/300)} dilution. Theresults presented in Table 13 suggest that there was a large range ofresponses in each group, i.e., the Coefficient of Variation (CV) rangedfrom 0.229 to 0.587, with the exception of the group immunized withvaccine containing MPL® where the CV was 0.051. Thus, it appeared thatMPL®, but not IL-12, may have acted as an adjuvant for the Pn14 IgGresponse and reduced the mouse-to-mouse variation. TABLE 12 Effect ofIL-12 on the IgG subclass response to Pn14 in mice immunized with abivalent PnPs6B/14 pneumococcal glycoconjugate vaccine PnPs14 Ig* PnPs14IgG Subclass Titer* at Week 5* Adjuvant Week 3 Week 5 IgG1 IgG2a IgG2bIgG3 200 ng IL-12 + 2,170 58,657 6,880 8,996 1,945 5,995 AlPO₄ 40 ng1,641 53,557 8,646 3,003 3,684 2,745 IL-12 + AlPO₄ 8 ng 2,181 85,17310,094 11,346 5,328 2,560 IL-12 + AlPO₄ AlPO₄ 2,102 201,082 54,989 4,0306,402 3,745 (no IL-12) 200 ng 849 18,293 5,769 1,582 536 799 IL-12 40 ng1,544 11,442 4,350 714 514 455 IL-12 8 ng IL-12 113 12,169 5,286 354 245330 None 509 22,601 6,080 808 618 694 100 μg 18,616 77,106 15,745 4,27510,205 3,916 MPL ®

[0076] TABLE 13 Response of individual mice to Pn14 component ofPn6B/Pn14 bivalent pneumococcal conjugate vaccine* O.D StandardCoefficient of Adjuvant O.D. Range Mean Deviation Variation AlPO₄ + 200ng IL-12 0.034-0.990 0.788 0.318 0.404 AlPO₄ + 40 ng IL-12 0.457-0.9480.771 0.176 0.229 AlPO₄ + 8 ng IL-12 0.023-0.923 0.707 0.281 0.397 AlPO₄(no IL-12) 0.328-0.974 0.770 0.220 0.285 8 ng IL-12 (no alum)0.009-0.812 0.505 0.292 0.587 No adjuvant 0.030-0.876 0.614 0.343 0.558100 μg MPL ^(®) 0.791-0.918 0.863 0.044 0.051

Example 5

[0077] Comparison of the Effect of IL-12 in the Presence or Absence ofAlum on the Murine Immune Response to Monovalent PnPs14-CRM₁₉₇ ConjugateVaccine

[0078] Study Design

[0079] BALB/c mice (8 per group) were immunized subcutaneously at week 0with 1 μg PnPs14-CRM₁₉₇ conjugate formulated with or without 100 μgAlPO₄ and either no IL-12 or with 8, 40, 200, 1,000 or 5,000 ng IL-12.Normal mouse serum (0.25%) was included as a carrier protein tostabilize IL-12 at low concentrations. At week 1, lymph node cellsuspensions were prepared from half the mice in each group and evaluatedfor antigen-specific cytokine production in vitro. Their spleens werealso harvested and weighed. At week 3 the remaining mice were bled andre-immunized with the same vaccine formulation used in the initialvaccination. At week 5 the twice-immunized mice were bled, their spleensweighed and their splenocytes evaluated for cytokine production. PnPs14and CRM₁₉₇ IgG and IgG subclass titers were determined on pooled sera.When the assays were performed using sera from individual mice, theresults are expressed as geometric mean titers (GMT).

[0080] Results

[0081] Effect of IL-12 on Spleen Weight One Week After Immunization

[0082] One week after the first immunization, mice receiving 5,000 ngIL-12, but not lower doses of IL-12, in the absence of AlPO₄, hadsignificantly higher spleen weights than those receiving vaccinecontaining neither alum nor IL-12 (Table 14). Vaccines containing AlPO₄induced higher spleen weights when formulated with 40 to 5000 ng IL-12.Pair-wise comparisons indicated that vaccines formulated with 200 or1000 ng IL-12 plus AlPO₄ induced higher spleen weights than thoseformulated with the same dose of IL-12 in the absence of AlPO₄. Overall,the data indicate that formulating IL-12 with AlPO₄ greatly enhanced abiological activity of the cytokine, i.e., its ability to causeincreased spleen weight one week after vaccination.

[0083] Effect of IL-12 on the IgG Response to PnPs14

[0084] Initially, pooled sera were assayed for IgG antibodies to PnPs14(Table 15). The clearest indication of an adjuvant effect was notedafter primary immunization with vaccine containing AlPO₄ and 8 to 40 ngIL-12. This combination resulted in a 17- to 21-fold increase in the IgGtiter relative to mice immunized with vaccine formulated with neitherAlPO₄ nor IL-12. The combination of AlPO₄ and IL-12 resulted in higherresponses than when used individually; on their own AlPO₄ and the 40 ngdose of IL-12 caused 4-fold and 5-fold increase in week 3 IgG titers,respectively. Analysis of individual sera from mice immunized withAlPO₄-containing vaccines (Table 16) showed that 8 ng IL-12 induced5-fold higher PnPs14 IgG titer after primary vaccination than vaccineadjuvanted with only AlPO₄. The difference in titers was statisticallysignificant. Higher doses of IL-12 did not enhance the response. The1,000 to 5,000 ng doses of IL-12 caused a marked decline in PnPs14 IgGtiters. After the second immunization only the 40 ng dose of IL-12caused a significant rise (3-fold) in the PnPs14 titer induced by theAlPO₄-based vaccine.

[0085] The pooled serum data suggest that the combination of AlPO₄ and8-40 ng IL-12 enhanced the IgG1 titers after primary immunization. Aftertwo vaccinations, IL-12 did not enhance the IgG1 titers to PnPs14 inmice immunized with conjugate in the absence of AlPO₄ as indicated byanalysis of pooled (Table 15) and individual sera (Table 17). Moreover,among mice immunized with vaccine containing AlPO₄, the addition of 8 to200 ng IL-12 did not result in higher IgG1 titers after 2 vaccinations(Table 17).

[0086] The most profound effect of IL-12 was to substantially increasethe PnPs14 IgG2a response at week 5. This was seen both when the vaccinecontained AlPO₄ or was formulated without AlPO₄ (Table 18). In theabsence of ALPO₄, statistically significant increases (14- to 42-fold)in IgG2a GMT were obtained with 8 to 1,000 ng IL-12. Similarly, 8-1,000ng IL-12 enhanced the ability of AlPO₄-containing vaccines to induceIgG2a antibodies, although in this study only the titers induced by the8 and 40 ng doses of IL-12 were statistically higher. Overall, thehighest IgG2a titers were induced by conjugate formulated with AlPO₄ and40 ng IL-12. This was significantly different from the IgG2a titersinduced by 40 ng IL-12 in the absence of AlPO₄, again indicating thatthe adjuvant activity of IL-12 was enhanced by alum.

[0087] IgG2b and IgG3 titers were assayed on pooled sera only (Table15). Doses of IL-12 in the range of 8 to 1,000 ng when co-formulatedwith AlPO₄, but not in its absence, promoted substantial increases inIgG3 titers after primary and secondary immunization. No consistenteffect of IL-12 on the IgG2b titers was noted.

[0088] Effect of IL-12 on the IgG Response to CRM₁₉₇

[0089] The IgG response to CRM₁₉₇ was also evaluated to see if therewere differences between the effect of IL-12 on the protein carrierversus the polysaccharide portion of the conjugate (Table 19). In theabsence of AlPO₄, 40 ng IL-12 appeared to modestly increase the IgGtiters to CRM₁₉₇ after two vaccinations. However, the highest IgG titersto CRM₁₉₇ were obtained when the vaccine was formulated with both AlPO₄and 8-40 ng IL-12. The heightened adjuvant activity of IL-12co-formulated with AlPO₄ is indicated by the finding that, on their own,40 ng IL-12 and AlPO₄ resulted in 6-fold and 17-fold increases in IgGtiter at week 5, but when combined together the increase was 147-fold.IL-12 enhanced the IgG1 response to CRM₁₉₇ regardless of whether thevaccine was formulated with or without AlPO₄ (Tables 19 and 20). IL-12substantially increased the week 5 IgG2a titers to CRM₁₉₇ afterimmunization with vaccines containing AlPO₄ (Table 19). Again theoptimal dose of IL-12 appeared to be 40 ng. The cytokine appeared toincrease the IgG2b titers induced by vaccine containing AlPO₄.

[0090] Effect of IL-12 on Cytokine Profile of CRM₁₉₇-Specific T Cells

[0091] Cytokine production by spleen cells taken two weeks aftersecondary vaccination (week 5) revealed effects of IL-12 on the primingof both IFN-γ and IL-5 producing cells. Splenocytes from mice immunizedin the absence of AlPO₄ and IL-12 produced detectable levels of IL-5,but not IFN-γ, when stimulated with CRM₁₉₇ in vitro (Table 21) .Formulating the vaccine with IL-12 appeared to enhance the induction ofIL-5-producing cells with peak activity occurring with 40 ng of thecytokine. Higher doses of IL-12 resulted in decreased production ofIL-5, with virtually no cytokine being produced by mice immunized withconjugate vaccine containing 1,000 to 5,000 ng IL-12. Convincing IFN-γproduction was detected only from the splenocytes of mice immunized withvaccines formulated with 5,000 ng IL-12. When the vaccine was formulatedwith AlPO₄, the addition of 8 ng IL-12 resulted in priming of cells thatproduced copious amounts of IFN-γ, whereas in the absence of thecytokine only antigen-specific IL-5 production was detected. It appearsthat priming for maximal IFN-γ production occurs with 40 to 1,000 ngIL-12. Addition of 5,000 ng IL-12 abolished the ability of the vaccineto prime for IL-5-producing cells. TABLE 14 Spleen weights of Balb/cmice one week after subcutaneous immunization with 1 μg PnPs14-CRM₁₉₇conjugate formulated with or without 100 μg AlPO₄ and the indicateddoses of IL-12 Spleen Weight (grams) Adjuvant Formulation Standard GroupCode IL-12 (ng) AlPO₄ AVERAGE Deviation P641 0 − 0.179 0.0225 P642 8 −0.148 0.0112 P643 40 − 0.162 0.0202 P644 200 − 0.175 0.0431 P645 1,000 −0.196 0.0068 P646 5,000 − 0.357 0.0247 P647 0 + 0.151 0.0158 P648 8 +0.151 0.0332 P649 40 + 0.217 0.0596 P650 200 + 0.290 0.0226 P651 1,000 +0.277 0.0919 P652 5,000 + 0.305 0.0545

[0092] Statistical Comparisons (ANOVA; α=0.05)

[0093] P642, P643, P644, P645 vs P641: not significant

[0094] P646 vs P641: significant

[0095] P648 vs P647: not significant

[0096] P649, P650, P651, P652 vs P647: significant

[0097] P641 vs P647: not significant

[0098] P644 vs P650: significant

[0099] P642 vs P648: not significant

[0100] P645 vs P651: significant

[0101] P643 vs P649: not significant

[0102] P646 vs P652: not significant TABLE 15 Anti-PnPs14 IgG responsein Balb/c mice immunized with PnPs14-CRM₁₉₇ conjugate formulated withIL-12 and AlPO₄ Adjuvant PnPs14 IgG Titers of Pooled Sera FormulationIgG IgG1 IgG2a IgG2b IgG3 IL-12 (ng) AlPO₄ Week 3 Week 5 Week 3 Week 5Week 3 Week 5 Week 3 Week 5 Week 3 Week 5 0 − 1,691 24,498 479 9,967 139492 <100 <100 295 1,516 8 − 4,679 32,966 841 9,860 377 1,902 108 609 3901,354 40 − 6,484 50,096 1,235 17,631 207 1,209 58 724 1,633 4,017 200 −5,330 51,240 385 7,568 715 3,748 290 1,397 1,091 4,519 1,000 − 6,34769,673 1,286 12,814 859 6,532 124 <100 782 6,208 5,000 − 1,131 19,621229 3,598 126 1,392 <100 <100 635 3,616 0 + 7,825 103,092 1,714 38,147195 1,535 617 3,973 447 2,963 8 + 29,506 195,069 7,444 58,046 1,2076,697 693 4,843 5,669 25,407 40 + 35,567 295,361 4,945 46,030 2,88317,267 1,371 9,911 5,797 22,602 200 + 10,177 190,701 1,777 41,800 6269,816 <100 1,479 3,443 23,648 1,000 + 2,422 245,683 90 31,373 167 13,847<100 722 1,173 34,039 5,000 + 1,304 35,333 91 5,228 <100 1,429 <100 <100772 8,065

[0103] TABLE 16 Effect of IL-12 on the IgG response to PnPs14 in miceimmunized with PnPs14-CRM₁₉₇ conjugate formulated with AlPO₄ PnPs14 IgGGMT (fold increase) Group IL-12 (ng) AlPO₄ Week 3 Week 5 P647 0 + 3,03727,027 P648 8 + 16,681 (5.5) 55,855 (2.1) P649 40 + 6,667 (2.2) 88,271(3.4) P650 200 + 2,333 (0.8) 57,076 (2.1) P651 1,000 + 611 (0.2) 30,886(1.1) P652 5,000 + 617 (0.2) 10,989 (0.4)

[0104] Statistical Comparisons (ANOVA; ∝=0.05)

[0105] Week 3 Titers

[0106] P648 vs P647: significant

[0107] P651 vs P647: significant

[0108] P649, P650, P652 vs P647: not significant

[0109] Week 5 Titers

[0110] P649 vs P647: significant

[0111] P648, P650, P651 vs P647: not significant TABLE 17 PnPs14 IgG1titers in mice twice immunized with PnPs14-CRM₁₉₇ conjugate vaccineformulated with or without AlPO₄ and various doses of IL-12 AdjuvantFormulation IgG1 GMT (Geometric Group Code IL-12 (ng) AlPO₄ Mean Titer)P641 0 − 9,492 P642 8 − 5,964 P643 40 − 14,028 P644 200 − 4,628 P6451,000 − 5,815 P646 5,000 − 1,757 P647 0 + 15,283 P648 8 + 35,730 P64940 + 31,855 P650 200 + 34,166 P651 1,000 + 15,347 P652 5,000 + 4,022

[0112] Statistical Comparisons (ANOVA; α=0.05)

[0113] P642, P643, P644, P645, P646, P647, P651 vs P641: not significant

[0114] P648, P649, P650 vs P641: significant

[0115] P648, P649, P650, P651 vs P647: not significant

[0116] P652 vs P647: significant TABLE 18 PnPs14 IgG2a titers in micetwice immunized with PnPs14-CRM₁₉₇ conjugate vaccine formulated with orwithout AlPO₄ and various doses of IL-12 IgG2a GMT at Week 5 Group CodeIL-12 (ng) AlPO₄ (Fold Increase*) P641 0 − 97 P642 8 − 1,418 (14.6) P64340 − 1,509 (15.6) P644 200 − 2,228 (23.0) P645 1,000 − 4,126 (42.5) P6465,000 − 289 (3.0) P647 0 + 806 P648 8 + 6,841 (8.5) P649 40 + 13,252(16.4) P650 200 + 4,740 (5.9) P651 1,000 + 3,291 (4.1) P652 5,000 + 368(0.5)

[0117] Statistical Comparisons (ANOVA; α=0.05)

[0118] P642, P643, P644, P645 vs P641: significant

[0119] P646 vs P641: not significant

[0120] P648, P649 vs P647: significant

[0121] P650, P651, P652 vs P647: not significant

[0122] P643 vs P649: significant TABLE 19 Anti-CRM₁₉₇ IgG response inBalb/c mice immunized with PnPs14-CRM₁₉₇ conjugate formulated with IL-12and AlPO₄ CRM₁₉₇ IgG Titer IgG Subclasses at Adjuvant (Pooled Sera) Week5 (Pooled Sera) IL-12 (ng) AlPO₄ Week 3 Week 5 IgG1 IgG2a IgG2b 0 −3,843 8,965 703 1,269 792 8 − 2,456 14,389 4,674 <100 <100 40 − 3,20053,758 14,073 3,403 <100 200 − 1,666 13,419 1,803 2,044 <100 1,000 −4,999 3,663 <100 506 <100 5,000 − 2,841 3,641 <100 <100 <100 0 + 4,870153,075 55,922 1,796 407 8 + 89,558 1,515,87 377,82 85,972 10,972 40 +19,566 1,319,10 147,03 199,29 7,206 200 + 6,884 315,071 48,852 36,8073,865 1,000 + 7,292 545,827 126,72 44,190 4,127 5,000 + 7,213 7,0291,041 769 <100

[0123] TABLE 20 IgG1 titers to CRM₁₉₇ in Balb/c mice immunized withPnPs14-CRM₁₉₇ conjugate formulated with IL-12 and AlPO₄ Group Code IL-12(ng) AlPO₄ IgG1 GMT Fold Increase P641 0 − 317 — P642 8 − 1,136 3.6 P64340 − 9,141 28.8  P644 200 − 1,627 5.1 P645 1,000 − 100 0.3 P646 5,000 −174 0.6 P647 0 + 22,061 — P648 8 + 119,130 5.4 P649 40 + 73,226 3.3 P650200 + 14,391 0.7 P651 1,000 + 33,468 1.5 P652 5,000 + 317  0.01

[0124] Statistical Comparisons (ANOVA; α=0.05)

[0125] P643, P644 vs P641: significant

[0126] P642, P645, P646 vs P641: not significant

[0127] P648, P649, P650, P651 vs P647: not significant

[0128] P642 vs P648, P643 vs P649, P644 vs P650, P645 vs P651:significant TABLE 21 Cytokine production by splenocytes from miceimmunized twice with PnPs14- CR₁₉₇ formulated with IL-12 in the presenceand absence of AlPO₄ Cells Stimulated IL-12 Dose in Vaccine FormulatedIL-12 Dose in Vaccine Formulated Cyto- With Without AlPO₄ (ng) WithAlPO₄ (ng) kine Antigen μg/mL 0 8 40 200 1,000 5,000 0 8 40 200 1,0005,000 IFN-γ CRM₁₉₇ 30 <0.06 <0.06 0.4 0.3 0.4 6.9 0.1 20.6 32.1 30.229.4 21.0 (U/mL) CRM₁₉₇ 10 <0.06 <0.06 0.3 0.2 0.3 3.4 0.1 16.5 31.730.2 27.6 21.0 CRM₁₉₇ 3 <0.06 <0.06 <0.06 <0.06 0.2 1.4 <0.03 15.7 30.928.7 26.7 20.1 CRM₁₉₇ 1 <0.06 <0.06 <0.06 <0.06 0.2 0.4 <0.03 12.8 30.928.2 27.9 18.0 CRM₁₉₇ 0.3 <0.06 <0.06 <0.06 <0.06 0.1 0.1 <0.03 6.3 27.526.2 26.5 6.5 Lysozyme 30 <0.06 <0.06 <0.06 <0.06 <0.03 <0.03 <0.03 <0.0<0.0 <0.0 <0.02 <0.02 Con A 1 11.9 15.4 15.8 15.9 20.6 21.0 21.1 17.020.8 13.7 23.4 17.7 Medium — <0.06 <0.06 <0.06 <0.06 <0.03 <0.03 <0.03<0.0 <0.0 <0.0 <0.02 <0.02 IL-5 CRM₁₉₇ 30 370 480 2560 960 60 70 20101440 5280 1640 880 <10 (pg/mL) CRM₁₉₇ 10 150 300 1110 260 <24 <24 1220920 4590 410 430 <10 CRM₁₉₇ 3 30 90 910 200 <24 <24 1190 690 2830 1030200 <10 CRM₁₉₇ 1 <4 50 200 40 <24 <24 880 400 2150 520 140 <10 CRM₁₉₇0.3 <4 30 70 <4 <24 <24 670 180 440 270 90 <10 Lysozyme 30 <4 <4 <4 <4<24 <24 <24 <24 <10 <10 <10 <10 Con A 1 <4 <4 <4 <4 <24 <24 60 80 40 <10<10 <10 Medium — <4 <4 <4 <4 <24 <24 <24 <24 <10 <10 <10 <10

Example 6

[0129] Effect of IL-12/AlPO₄ on the Humoral Response to a NonavalentPneumococcal Glycoconjugate Vaccine

[0130] Study Design

[0131] Evaluation of the effect of IL-12 on the IgG response topneumococcal glycoconjugate vaccine was extended to a nonavalent vaccinecomposed of serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F. SwissWebster mice were immunized with 0.1, 1, or 5 μg of vaccine(carbohydrate weight) at weeks 0 and 3. The vaccine was administeredalone, with AlPO₄ (100 μg) or with AlPO₄ admixed with 50, 200 or 1,000ng of IL-12. Normal mouse serum was not included in the vaccine. The IgGresponses to serotypes 4, 6B, 9V, 14, 18C and to the carrier proteinCRM₁₉₇ were evaluated at week 5 (i.e., 2 weeks after boosting) by ELISA.

[0132] Results

[0133] Response to CRM₁₉₇ at Week 5

[0134] Addition of IL-12 to a vaccine containing AlP04 resulted in adose-dependent increase in IgG2a and IgG2b antibodies to CRM₁₉₇. Thiswas seen at all doses of conjugate tested (Table 22). Increased IgG2atiters were evident in mice receiving 50 ng of the cytokine and weremaximal at 1,000 ng. This contrasts with other studies where maximalIgG2a titers were obtained with 40-100 ng of cytokine added to thealum-based vaccine and where higher doses of IL-12 resulted in adiminished immune response. The reason for the differences in doseresponse between studies is not known. It may relate to differences inthe vaccine, i.e., multivalent versus monovalent or that normal mouseserum included in the vaccine in previous studies to stabilize thecytokine at low concentrations was omitted.

[0135] Response to Pneumococcal Polysaccharides

[0136] Formulating the nonavalent vaccine with AlP04 enhanced the IgGresponse to several serotypes including PnPs4, PnPs6B, PnPs9V andPnPs14, especially when the lowest dose of conjugate (0.1 μg) was used(Tables 24-27). Addition of IL-12 did not appear to further enhance theIgG response to these serotypes. In the case of the PnPs18C response,however, addition of 50 or 1,000 ng IL-12 to 5 μg of vaccine containingAlP04 resulted in higher geometric mean IgG titers to this serotype andhigher proportion of mice with PnPs18C IgG titers above 10,000 (Table23). The responses to PnPs1, 5, 19F and 23F were not evaluated.

[0137] Addition of IL-12 to nonavalent vaccines containing AlP04resulted in dose-dependent increases in IgG2a titers to PnPs4, PnPs6B,PnPs9V and PnPs14 (Tables 24-27). Generally, the increase in IgG2aparalleled that for the CRM₁₉₇ response with highest titers beingobtained with 1,000 ng of IL-12. In contrast to the experiments usingmonovalent PnPs14 conjugate or bivalent PnPs6B/PnPs14 vaccine, the 50 ngdose of IL-12 had little or no effect on the IgG2a response to theseserotypes. The exception is the IgG2a response to PnPs14, as this doseof cytokine appeared to enhance the response to this serotype (Table27).

[0138] Overall, this study indicates that IL-12 will promote thecomplement-fixing IgG2a antibody subclass response to multiplepneumococcal serotypes present in a multivalent vaccine. TABLE 22 Effectof IL-12 on the CRM₁₉₇ response of mice immunized with nonavalentpneumococcal glycoconjugate vaccine formulated with AlP0₄ VaccineFormulation Con- ju- gate Dose IL-12 A1P04 CRM₁₉₇ Response at Week 5(μg) (ng) (μg) IgG IgG1 IgG2a IgG2b IgG3 5.0 none none 391,021 138,7841,687 3,277 102 5.0 0 100 1,419,910 609,704 4,328 11,349 181 5.0 50 1003,226,410 896,621 27,736 18,086 728 5.0 200 100 2,991,990 584,991 87,73228,855 2,937 5.0 1,000 100 16,224,900 906,192 303,656 87,726 3,023 1.0none none 545,046 162,757 1,178 9,213 358 1.0 0 100 956,584 338,7511,284 4,118 306 1.0 50 100 1,936,170 370,961 6,677 31,982 931 1.0 200100 4,788,500 660,082 187,034 36,785 1,065 1.0 1,000 100 12,404,500644,151 533,065 69,185 1,176 0.1 none none 15,215 3,800 <100 <100 <1000.1 0 100 561,952 157,362 1,437 7,744 <100 0.1 50 100 807,363 141,67016,064 26,978 2,092 0.1 200 100 1,560,380 313,263 38,686 51,737 306 0.11,000 100 2,296,310 202,111 112,158 36,958 1,054

[0139] Mice were immunized with the indicated dose of nonavalentpneumococcal glycoconjugate vaccine at weeks 0 and 3. The conjugateswere formulated alone, with AlPO₄ (100 μg) or with AlPO₄ plus IL-12.Sera from the week 5 bleed were analyzed for IgG antibodies to CRM₁₉₇.TABLE 23 Effect of IL-12 on the response to PnPs18C in mice immunizedwith 5 μg nonavalent pneumococcal glycoconjugate vaccine formulated withAlP04 Vaccine Formulation PnPs18C Response Conjugate IL-12 AlP0₄ IgGTiter Mice With Titer Dose (μg) (ng) (μg) (GMT) >10,000 (n = 5) 5 0 02,933 1 5 50 100 23,725 4 5 1,000 100 48,375 5

[0140] Mice were immunized with the indicated dose of nonavalentpneumococcal glycoconjugate vaccine at weeks 0 and 3. The conjugateswere formulated alone, with AlPO₄ (100 μg) or with AlPO₄ plus IL-12.Individual mouse sera from the week 5 bleed were analyzed for IgGantibodies to PnPs18C. TABLE 24 Effect of IL-12 on the PnPs4 response ofmice immunized with nonavalent pneumococcal glycoconjugate vaccineformulated with AlP04 Vaccine Formulation Con- ju- gate IL-12 AlP0₄PnPs4 Response at Week 5 Dose (μg) (ng) (μg) IgG IgG1 IgG2a IgG2b IgG35.0 none none 55,068 13,731 <500 <500 <500 5.0 0 100 233,008 55,620 <5001,157 990 5.0 50 100 285,806 64,493 1,050 1,329 2,634 5.0 200 100203,236 56,654 1,789 692 2,693 5.0 1,000 100 371,329 35,778 4,048 1,0803,820 1.0 none none 77,714 9,070 <500 608 <500 1.0 0 100 141,371 14,829<500 <500 542 1.0 50 100 97,999 14,336 449 814 1,034 1.0 200 100 137,67417,380 752 569 816 1.0 1,000 100 214,739 25,056 4,685 1,260 4,055 0.1none none 4,726 706 <500 <500 <500 0.1 0 100 79,686 12,071 <500 869 <5000.1 50 100 70,917 9,649 1,032 1,389 <500 0.1 200 100 46,503 7,799 8851,056 572 0.1 1,000 100 87,762 6,788 1,725 <500 1,682

[0141] Mice were immunized with the indicated dose of nonavalentpneumococcal glycoconjugate vaccine at weeks 0 and 3. The conjugateswere formulated alone, with AlP04 (100 μg) or with AlP04 plus IL-12.Sera from the week 5 bleed were analyzed for IgG antibodies to PnPs4.TABLE 25 Effect of IL-12 on the PnPs6B response of mice immunized withnonavalent pneumococcal glycoconjugate vaccine formulated with AlP04Vaccine Formulation Con- jugate PnPs6B Response at Week 5 Dose IL-12AlPO₄ Total (μg) (ng) (μg) IgG IgG1 IgG2a IgG2b IgG3 5.0 none none64,734 20,221 <100 195 325 5.0 0 100 103,686 39,061 138 2,498 1,801 5.050 100 487,798 127,753 916 3,200 13,758 5.0 200 100 214,743 59,979 924959 6,459 5.0 1,000 100 427,514 94,478 4,426 2,552 13,142 1.0 none none165,588 37,646 <100 2,047 2,337 1.0 0 100 730,920 133,441 990 2,7707,468 1.0 50 100 428,549 77,124 838 3,755 12,931 1.0 200 100 164,82029,685 316 662 4,703 1.0 1,000 100 401,513 51,132 11,442 2,735 31,6130.1 none none 4,787 1,034 <100 <100 <100 0.1 0 100 370,177 71,287 60311,372 5,712 0.1 50 100 137,091 25,447 1,029 3,346 3,411 0.1 200 100128,428 31,634 434 2,698 1,891 0.1 1,000 100 524,385 67,301 9,611 11,5878,711

[0142] Mice were immunized with the indicated dose of nonavalentpneumococcal glycoconjugate vaccine at weeks 0 and 3. The conjugateswere formulated alone, with AlP04 (100 μg) or with AlP04 plus IL-12.Sera from the week 5 bleed were analyzed for IgG antibodies to PnPs6B.TABLE 26 Effect of IL-12 on the PnPs9V response of mice immunized withnonavalent pneumococcal glyconconjugate vaccine formulated with AlP04Vaccine Formulation PnPs9V Response at Week 5 Conjugate IL-12 AlPO₄Total Dose (μg) (ng) (μg) IgG IgG1 IgG2a IgG2b IgG3 5.0 none none 36,83115,568 306 250 317 5.0 0 100 78,614 37,544 359 667 286 5.0 50 100117,345 61,031 1,073 834 2,089 5.0 200 100 134,333 35,031 2,973 7482,594 5.0 1,000 100 197,407 40,368 15,353 2,147 1,945 1.0 none none81,932 34,845 546 2,232 735 1.0 0 100 100,448 55,608 660 1,274 699 1.050 100 157,316 47,285 1,084 2,036 4,730 1.0 200 100 154,672 48,318 1,765860 2,044 1.0 1,000 100 168,614 54,223 10,037 1,469 3,006 0.1 none none<500 181 <100 <100 <100 0.1 0 100 86,952 26,425 206 485 1,285 0.1 50 10020,746 6,381 579 726 353 0.1 200 100 19,966 5,501 778 325 235 0.1 1,000100 50,219 3,511 1,290 1,036 714

[0143] Mice were immunized with the indicated dose of nonavalentpneumococcal glycoconjugate vaccine at weeks 0 and 3. The conjugateswere formulated alone, with AlP04 (100 μg) or with a AlP04 plus IL-12.Sera from the week 5 bleed were analyzed for IgG antibodies to PnPs9V.TABLE 27 Effect of IL-12 on the PnPs14 response of mice immunized withnonavalent pneumococcal glyconconjugate vaccine formulated with AlP04Vaccine Formulation Conju- gate PnPs14 Response at Week 5 Dose IL-12AlPO₄ Total (μg) (ng) (μg) IgG IgG1 IgG2a IgG2b IgG3 5.0 none none 2,6761,750 <100 <100 <100 5.0 0 100 11,792 15,704 124 580 1,723 5.0 50 10056,712 31,056 6,144 2,854 11,840 5.0 200 100 5,049 3,050 1,588 <1002,106 5.0 1,000 100 11,848 3,760 1,853 366 2,035 1.0 none none 4,8463,116 <100 409 699 1.0 0 100 20,605 31,022 291 2,383 9,286 1.0 50 1008,338 4,722 1,354 715 10,079 1.0 200 100 5,618 3,252 1,014 <100 583 1.01,000 100 13,026 3,551 2,879 671 2,070 0.1 none none <100 105 <100 <100<100 0.1 0 100 114 392 <100 <100 710 0.1 50 100 2,140 2,838 <100 2453,592 0.1 200 100 2,200 426 <100 622 759 0.1 1,000 100 394 378 219 100658

[0144] Mice were immunized with the indicated dose of nonavalentpneumococcal glycoconjugate vaccine at weeks 0 and 3. The conjugateswere formulated alone, with AlP04 (100 μg) or with AlP04 plus IL-12.Sera from the week 5 bleed were analyzed for IgG antibodies to PnPs14.

Example 7

[0145] The effect of IL-12 and AlP04 on the Immune Response to Neiserriameningitidis Type C (menC) Glyconconjugate Vaccine

[0146] Study Design

[0147] This study evaluated IL-12 with a vaccine against Neiserriameningitidis type C (menC). Swiss Webster mice were immunized at weeks 0and 3 with 0.1 μg or 1 μg of MenC glycoconjugate formulated alone, withAlPO₄ (100 μg) or a combination of IL-12 (50 ng) and AlP04. Normal mouseserum was not added to the vaccine. Mice were bled at weeks 3 and 5 andsera analyzed for IgG antibodies to menC polysaccharide by ELISA.

[0148] Results

[0149] When immunized with the higher dose of conjugate, equivalent menCIgG titers were generated regardless of the adjuvant formulation. Theaddition of IL-12/AlP04 to the vaccine, however, resulted in higherIgG2a titers to the polysaccharide than if formulated with AlP04 (but noIL-12) or no adjuvant.

[0150] In mice immunized with the lower dose of conjugate, higher meningC titers were obtained when the vaccine was formulated with AlP04 (Table28). The addition of IL-12 to the adjuvant did not enhance the overallIgG titer but did result in a >10-fold increase in IgG2a antibodies.These data show that IL-12 in combination with AlP04 can promote theinduction of complement-fixing IgG subclasses to menC glyconconjugatevaccine. TABLE 28 Effect of IL-12/AlP04 on the IgG response to menCglyconconjugate vaccine Vaccine Formulation MenC Response MenC ConjugateIL-12 AlPO₄ IgG IgG Subclass at Week 5 (μg) (ng) (μg) Week 3 Week 5 IgG1IgG2a IgG2b IgG3 1.0 50 100 33,176 598,027 83,662 7,218 4,351 1,436 0100 34,553 404,111 71,017 1,383 3,085 1,006 0 0 16,254 288,493 63,0431,965 <100 502 0.1 50 100 2,584 68,678 9,604 3,440 1,967 512 0 100 8,17430,450 6,532 288 429 <100 0 0 1,724 7,894 1,767 <100 <100 <100

Example 8

[0151] The effect of IL-12 and AlP04 on the Immune Response toHemophilus influenzae Type b Glyconconjugate Vaccine (HbOC)

[0152] Study Design

[0153] This study evaluated IL-12 with a vaccine against Hemophilusinfluenzae type b. Swiss Webster mice (10 per group) were immunized atweeks 0 and 3 with 0.1 μg or 1.0 μg of glyconconjugate vaccineconsisting of capsular polysaccharide from Hemophilus influenzae type b(HibPs) conjugated to CRM₁₉₇. The vaccine (HbOC) was administered aloneor in combination with AlP04 (100 μg) or a mixture of IL-12 (50 ng) plusAlP04. Normal mouse serum was not added to the vaccine. The mice werebled at weeks 3 and 5. The antibody response to HibPs was measured usinga Farr assay which measures all antibodies binding to the saccharideregardless of isotype, i.e., IgM, IgG and IgA. The IgG subclass responsewas measured by ELISA. Additionally, the IgG and IgG subclass responseto CRM₁₉₇ was also determined by ELISA.

[0154] Results

[0155] The titers of anti-HibPs antibodies in serum pooled from the week3 bleed (primary response) were not different between mice immunizedwith vaccine formulated alone, with AlP04 or IL-12 plus AlP04 regardlessof the dose of conjugate used for immunization (Table 29). Analysis ofpooled serum from the week 5 bleed suggested that in mice immunized with1 μg of HbOC with IL-12 plus alum resulted in at least a 10-fold higheranti-HibPs than when given with alum or without adjuvant (Table 30).However, analysis of individual mouse sera showed that this was due to asingle mouse having a titer of approximately 10,000 μg/mL. When theresults are expressed as geometric mean titer there was no evidence ofan enhanced HibPs response due to IL-12. The IgG subclass response toHibPs was evaluated on pooled sera by ELISA. The combination of IL-12and AlP04 appeared to enhance the IgG2a titer 3-fold in mice immunizedwith 1 μg of conjugate. However, this was no different than the titerobtained with vaccine adjuvanted with AlP04 alone. In mice immunizedwith 0.1 μg of HbOC, IL-12 plus AlP04 did not enhance the IgG2a titer toHibPs. That the IL-12/AlP04 adjuvant combination was active was revealedby analysis of the anti-CRM₁₉₇ response (Table 31) where increased IgG2atiter to the carrier protein was seen in mice immunized with either doseof conjugate. TABLE 29 Anti-HibPs antibody response of mice immunizedwith HbOC formulated with IL-12 and AlP04 Anti-HibPs Antibody Response(μg/mL) Vaccine Formulation Week 3 Week 5 HbOC IL-12 AlPO₄ Pooled Pooled(μg) (ng) (μg) Serum Serum GMT* 1.0 50 100 9.73 469.16 26.92 0 100 10.0442.55 21.30 0 0 5.12 33.19  2.25 0.1 50 100 3.18 30.95 ND 0 100 4.0615.11 ND 0 0 3.03 14.05 ND

[0156] TABLE 30 Effect of IL-12 and AlP04 on the IgG subclass responseto HbOC Anti-HibPs IgG Subclass Response at Week 5 (ELISA VaccineFormulation Endpoint Titer) HbOC (μg) IL-12 (ng) AlPO_(4 (μg)) IgG1IgG2a 1.0 50 100 754,745 26,899 0 100 122,637 12,880 0 0 73,114 8,5700.1 50 100 46,673 17,290 0 100 68,176 14,971 0 0 35,237 11,418

[0157] TABLE 31 Anti-CRM₁₉₇ IgG response of mice immunized with HbOCformulated with IL-12 and AlP04 Vaccine Formulation HbOC IL-12 AlPO₄Anti-CRM₁₉₇ Response at Week 5 (μg) (ng) (μg) IgG IgG1 IgG2a IgG2b 1.050 100 1,775,700 681,944 39,672 40,527 0 100 2,221,780 818,557 19,01032,672 0 0 3,979,530 1,466,010 8,059 15,961 0.1 50 100 761,027 292,44838,258 21,008 0 100 891,251 346,728 6,546 14,832 0 0 874,805 151,3971,899 3,517

[0158] Equivalents

[0159] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

We claim:
 1. A composition comprising a mixture of a meningococcalcapsular polysaccharide, an adjuvant amount of interleukin-12 and anaqueous suspension of alum, and optionally comprising a physiologicallyacceptable vehicle.
 2. A composition according to claim 1, wherein theinterleukin-12 is adsorbed onto the alum suspension.
 3. A compositionaccording to claim 1, wherein the interleukin-12 is humaninterleukin-12.
 4. A composition according to claim 1, wherein the alumis aluminum hydroxide or aluminum phosphate.
 5. A composition accordingto claim 1, wherein the meningococcal capsular polysaccharide is theNeisseria meningitidis type C capsular polysaccharide.
 6. A compositionaccording to claim 1, wherein the meningococcal capsular polysaccharideis conjugated to a carrier molecule.
 7. A composition according to claim6, wherein the carrier molecule is selected from the group consisting oftetanus toxin, diphtheria toxin, pertussis toxin and non-toxic variantsthereof.
 8. A composition according to claim 7, wherein the carriermolecule is CRM₁₉₇.
 9. A method of eliciting an immune response to ameningococcal capsular polysaccharide, comprising administering to amammalian host an effective amount of a vaccine composition comprising amixture of a meningococcal capsular polysaccharide, an adjuvant amountof interleukin-12 and an aqueous suspension of alum, and optionallycomprising a physiologically acceptable vehicle.
 10. A method accordingto claim 9, wherein the interleukin-12 is adsorbed onto the alumsuspension.
 11. A method according to claim 9, wherein theinterleukin-12 is human interleukin-12.
 12. A method according to claim9, wherein the alum is aluminum hydroxide or aluminum phosphate.
 13. Amethod according to claim 9, wherein the meningococcal capsularpolysaccharide is the Neisseria meningitidis type C capsularpolysaccharide.
 14. A method according to claim 9, wherein themeningococcal capsular polysaccharide is conjugated to a carriermolecule.
 15. A method according to claim 14, wherein the carriermolecule is selected from the group consisting of tetanus toxin,diphtheria toxin, pertussis toxin and non-toxic variants thereof.
 16. Amethod according to claim 15, wherein the carrier molecule is CRM₁₉₇.17. An immunogenic composition comprising a mixture of a meningococcalcapsular polysaccharide, an adjuvant amount of interleukin-12 and anaqueous suspension of alum, and optionally comprising a physiologicallyacceptable vehicle.
 18. An immunogenic composition according to claim17, wherein the interleukin-12 is adsorbed onto the alum suspension. 19.An immunogenic composition according to claim 17, wherein theinterleukin-12 is human interleukin-12.
 20. An immunogenic compositionaccording to claim 17, wherein the alum is aluminum hydroxide oraluminum phosphate.
 21. An immunogenic composition according to claim17, wherein the meningococcal capsular polysaccharide is the Neisseriameningitidis type C capsular polysaccharide.
 22. An immunogeniccomposition according to claim 17, wherein the meningococcal capsularpolysaccharide is conjugated to a carrier molecule.
 23. An immunogeniccomposition according to claim 22, wherein the carrier molecule isselected from the group consisting of tetanus toxin, diphtheria toxin,pertussis toxin and non-toxic variants thereof.
 24. An immunogeniccomposition according to claim 23, wherein the carrier molecule isCRM₁₉₇.